A widely recognized limitation of MRI cell tracking using nanoparticle probes is that they cannot report on cellular activity or cell viability in vivo Non-invasive cell tracking methods that can monitor changes in cell viability and activation would be a great value in the development of cancer treatments, cell therapies, and anti-inflammatory drugs. Towards these goals, we will explore the utility of real-time monitoring of intracellular oximetry in vivo using perfluorocarbon (PFC) emulsion imaging probes. This proposal builds on our prior work developing MRI cell tracking methods for ex vivo and in situ labeling of cells with PFC imaging reagents. For ex vivo cell labeling, isolated cells of interest (e.g., leukocytes, stem cells, or cancer cells) are labeled in culture with PFC emulsion, and following transfer to the subject, cells are tracked in vivo using fluorine-19 (19F) MRI. The fluorine signal yields cell-specific images, with no background, that can be used to quantify apparent cell numbers at sites of accumulation. For in situ labeling, PFC emulsion is injected intravenously and taken up by macrophages that home to sites of inflammation and can be visualized by 19F MRI. In this proposal, we will exploit the oxygen sensing properties of the intracellular PFC molecules. Oxygen binding to PFC results in a reduction in the 19F spin-lattice relaxation time (T1), where T1 varies linearly with the partial pressure of oxygen (pO2). Hence, we propose combining 19F-based cell tracking with 19F T1 measurements to monitor intracellular pO2 in a cell-specific manner. Results from our lab in cancer models have demonstrated the feasibility of measuring the absolute intracellular pO2 in tumor cells and response to anti-cancer treatments. Overall, the proposal has two Specific Aims. Aim 1: Can intracellular pO2 detect cell viability in vivo? We will test the hypothesis that a measureable increase in pO2 is an indirect consequence of apoptotic processes. In tumor cells, we will characterize the cellular pO2 response following chemotherapy, suicide gene therapy, and effector T cell immunotherapy, all of which drive the cell towards apoptosis, but employ different triggers. The establishment of a relationship between cell viability and intracellular pO2 can be exploited in the preclinical evaluation of emerging cancer therapies. Moreover, as human clinical translation of 19F cell tracking matures, these oximetry techniques may be used in cell therapy clinical trials to determine whether the cellular graft is viable after delivery to the patient. Stdies in tumor models will help set the foundation for future studies in a variety of therapeutic cell types. Aim 2: Can macrophage intracellular pO2 detect immunoactivity? In a further extension, we will test the hypothesis that macrophages localized at sites of inflammation will respond to pharmacological immunosuppression by altering intracellular pO2 levels. A murine model of inflammatory bowel disease will be used, along with in situ PFC labeling of macrophages. We will test whether macrophage intracellular pO2 can be used as a tool to assess anti-inflammatory drugs non-invasively. Overall, the proposed experiments will set the foundation for a broad field of inquiries using intracellular PFC cell tracking agents to garner information about real-time cell metabolism in vivo.